The receptor-mediated actions of gonadotropin-releasing hormone (GnRH) and other regulators of pituitary hormone secretion are studied in cultured anterior pituitary cells. Occupancy of pituitary receptors and activation of gonadotropin secretion was followed by receptor turnover and subsequent up-regulation of receptor number. In contrast, potent GnRH antagonists dissociated slowly following receptor binding and did not cause down-regulation of receptors and desensitization of the gonadotroph. The bound antagonist remained localized at the cell surface for much longer periods than bound agonist analogs, suggesting that receptor activation is necessary to initiate internalization of the hormone-receptor complex. The ability of GnRH to enhance phosphatidylinositol (PI) turnover with increased formation of phosphatidic acid (PA) and arachidonic acid (AA) led to studies on the actions of these compounds on LH release. PA stimulated cGMP formation and LH release in a calcium-dependent manner, suggesting that endogenous PA may function as a calcium ionophore in the gonadotropic action of GnRH and its agonist analogs. Protein kinase C was shown to be abundant in purified gonadotrophs and to be activated by phorbol esters and diacylglycerols, which also stimulated LH release. The distribution of protein kinase C between cytosol and membrane fractions was analyzed in cultured pituitary gonadotrophs during treatment with GnRH. GnRH-stimulated release of luteinizing hormone (LH) from gonadotroph-enriched cells was accompanied by a rapid and dose-dependent decrease in cytosolic protein kinase C and by a corresponding increase in the particulate enzyme. Retinal directly inhibited the activity of cytosolic protein kinase C and also attenuated the release of LH from GnRH-stimulated gonadotrophs. These findings, and the ability of GnRH to cause rapid translocation of cytosolic protein kinase C to a membrane-associated form, suggest that hormonal activation of protein kinase C is an intermediate step in the stimulation of pituitary LH secretion by GnRH.